This two-part project studies the organization and function of specialized membranes in neurons and glia. The first part aims to characterize calcium regulation of synaptic activity in parallel fiber/Purkinje cell synapses of the cerebellar cortex. Major advances in frozen sectioning technology and the application of a new analytical instrument-the low-temperature, high-resolution, field-emission scanning transmission electron microscope (described in Project Z01 -NS-02836-01 LN)- have permitted a detailed re-investigation by energy-dispersive x-ray microanalysis (EDX) of the role of endoplasmic reticulum (ER) in regulating intracellular calcium in these synapses. Extending the previous study, the results show that, as a consequence of accumulating and releasing calcium during synaptic activity, the calcium content of the ER in dendritic spines cycles between two characteristic concentration states. The calcium concentration of ER in terminal dendrites changes even more dramatically during activity, but the pattern is more complex. Preliminary EDX analysis of cryosections from new, in vitro preparations of cerebellum, as well as structural analysis of new organotypic culture of hippocampal slices, indicate that it is possible to analyze synapses and dendrites which are in pharmacologically defined states. In Part 2, the formation of specialized membranes is studied in the context of myelin assembly. Oligodendrocytes and Schwann cells use distinct pathways for synthesis, sorting, transport and assembly of different myelin-specific proteins; some of these pathways appear to depend on microtubule (MT)-based intracellular transport. Confocal light microscopy shows that the perinuclear region of myelinating Schwann cells contain a principal, acentrically placed Golgi apparatus associated with one microtubule organizing center and a major MT network. This arrangement contrasts with the MT bundles in the internodes of the Schwann cell, where a significant fraction (approx. 30%) of these bundles are assembled with the (+)-end directed toward the soma. Immunogold electron microscopy shows that from 2-35 days of development, the MTs of the internode are not sorted into domains on the basis of microtubule-associated protein binding or post-translational modification (acetylation and tyrosination).